Opioid receptors undergo rapid, ligand-dependent endocytosis by clathrin-coated pits. Mounting evidence implicates this process as a fundamental component of the complex physiological regulation of opioid receptors, which is induced by native opioid peptides and distinguishes the actions of addictive opiate drugs. Therefore, understanding molecular mechanisms that control this process has broad relevance to opioid biology and addiction research. One mechanism by which ligands control the entry of opioid receptors into clathrin-coated pits is mediated by the phosphorylation-dependent association of agonist-activated receptors with beta-arrestin. Preliminary studies suggest the existence of an additional mechanism of endocytic regulation, which is expressed in a cell type-specific manner and appears to be mediated by novel protein interactions. The studies proposed in this application are designed to identify specific proteins that mediate this regulatory mechanism and examine the function of these proteins in several cell types. The Specific Aims of the proposed studies are to (1) define cytoplasmic residues required for cell type-specific endocytosis of a truncated mutant opioid receptor, (2) identify and clone cellular proteins which interact with the defined receptor domain(s), and (3) elucidate the functional role of selected interacting proteins in regulating opioid receptor endocytosis. These studies will contribute to a fundamental molecular understanding of mechanisms that regulate opioid receptors and mediate cell type-specific differences in receptor regulation. Thus the proposed research is directly relevant to understanding the cellular physiology of opiate drug action and addiction in complex neural tissue.